Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli

Abstract

katAencodes the major catalase that accounts for 90 % of the total catalase activity present inXanthomonas campestrispv.phaseoli.katAis located upstream of an ORF designatedankAencoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis ofkatAandankAidentified twokatAtranscripts: a major monocistronickatAtranscript and a minor bicistronickatA–ankAtranscript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of thekatApromoter region showed a putative OxyR binding site located upstream of anEscherichia coli-likeσ70−35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to thekatApromoter. AkatAmutant was highly sensitive to H2O2during both the exponential and stationary phases of growth. This phenotype could be complemented by functionalkatA, confirming the essential role of the gene in protectingX. campestrisfrom H2O2toxicity. Unexpectedly, inactivation ofankAalso significantly reduced resistance to H2O2and the phenotype could be complemented by plasmid-borne expression ofankA. Physiological analyses showed thatkatAplays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2killing inX. campestris.